New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants.
Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20mM sodium phosphate buffer pH 7.5, containing 0.1M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn(2+)-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.
Platis
D
D
Agricultural University of Athens, Department of Agricultural Biotechnology, 75 Iera Odos, GR 118 55 Athens, Greece.
Drossard
J
J
Fischer
R
R
Ma
J K-C
JK
Labrou
N E
NE
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000080908:Broadly Neutralizing Antibodies; D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000191:economics / Q000379:methods; D015483:HIV Antibodies / Q000302:isolation & purification*; D006801:Humans; D008670:Metals / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000737:chemistry* / Q000276:immunology; D011786:Quality Control; D014026:Nicotiana / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
C529157:2G12 monoclonal antibody; D000911:Antibodies, Monoclonal; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D008670:Metals